ES2534769T3 - Diagnosis of B lymphocyte lymphoma - Google Patents

Abstract

A procedure for the diagnosis of a B lymphocyte lymphoma, characterized by the detection of genetic aberrations of the NAV3 gene in a biological sample, indicating the presence of aberrations a B lymphocyte lymphoma.

Description

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DESCRIPTION

Diagnosis of B lymphocyte lymphoma

Field of the Invention

The present invention relates to the fields of genetics and oncology and provides procedures and means for the diagnosis and monitoring of patients who have B lymphocyte lymphomas, such procedures and means allow early diagnosis of B lymphocyte lymphoma. Specifically the invention. It refers to a new procedure and a biomarker to diagnose B lymphocyte lymphomas and to differentiate B lymphocyte lymphomas into prognosis groups of indolent and aggressive B lymphocytes.

Background of the invention

The current concept of cancer development includes the development of chromosomal instability, aneuploidy and a series of acquired genetic aberrations that affect genes important for cell growth and survival. It has been suggested that stem cells have a critical role in the development of cancer, and that epigenetic regulation is relevant to the genetic expression associated with pluripotency. Also, unique genetic aberrations can influence the entire pathogenesis process, and they can also be useful as cancer markers. During the past years, genetic aberrations acquired in several cancers (for example HER / neu in breast cancer) have been found and molecular cytogenetic assays based on these genes are on the market and are routinely used in the diagnosis of cancer in the clinical laboratories

Lymphomas are cancers of lymphoid tissue. They comprise a group of heterogeneous cancers, divided into non-Hodgkin lymphomas (NHL) and Hodgkin lymphomas (HL). There are more than 40 subgroups, depending on the type and maturity of the underlying malignant lymphoid cell. NHL usually originates in lymphoid tissues and can be classified as T-lymphocyte lymphoma or B-lymphocyte. Most NHLs (i.e. 80-90%) are of B-lymphocyte origin. Non-Hodgkin B-cell lymphomas they include Burkitt lymphoma, diffuse large B lymphocyte lymphoma, follicular lymphoma, large immunoblastic cell lymphoma, precursor B lymphoblastic lymphoma, and mantle cell lymphoma. Non-Hodgkin T-cell lymphomas include fungal mycoses, large anaplastic cell lymphoma, and precursor T lymphoblastic lymphoma. Lymphomas related to lymphoproliferative disorders after bone marrow transplantation or stem cells are usually non-Hodgkin B-cell lymphomas.

The prognosis of NHL depends on the histological type, stage, and treatment. Based on these variables, NHLs can be divided into two prognostic groups: indolent lymphomas and aggressive lymphomas. The types of indolent NHL lymphoma have a relatively good prognosis but are not curable in advanced stages. Most indolent types are of nodular (or follicular) morphology. Follicular lymphoma (FL) is by far the most common of indolent NHLs and represents almost 25% of all new cases of NHL. The type of aggressive NHL (for example diffuse large B lymphocyte lymphoma (DLBCL)) has a shorter life expectancy. With the modern treatment of patients with NHL, total survival at 5 years is approximately 50% to 60%. Of patients with aggressive NHL, 30% to 60% can be cured but the vast majority of relapses occur in the first 2 years after therapy.

The incidence of different lymphomas increases continuously and is currently around 363,000 new cases annually worldwide.

In the treatment of lymphomas, an early diagnosis is crucial, because the disease tends to recur in later stages. In the diagnosis of lymphoma, a biopsy of a lymph node is obtained and a histopathological analysis is performed. The type of lymphoma can be identified by the physical appearance of cancer cells under the microscope or by the use of markers that identify special molecules of lymphoma cells. Follicular lymphoma graduation is based on the average number of large cells transformed into 10 neoplastic follicles in a high-power examination field at x10-40. The reproducibility of follicular lymphoma graduation depends on the experience of the observer; therefore, significant variations occur.

Currently, there are no molecular biological biomarkers available in the clinical setting to identify patients with NHL as soon as possible, or to identify patients in whom malignant cell clones persist despite a clinically effective therapy.

Chromosomal aberrations are frequently seen in lymphomas and quite often such aberrations occur in the 12q region (Bea et al., 1996, Benz et al., 1996, Horsman et al., 2001, Hernandez et al., 2001, Lestou and col., 2003, Chui et al., 2003, Hallerman et al., 2004). The most common chromosomal abnormality associated with NHL is the translocation t (14; 18) (q32; q21) found in 85% of follicular lymphomas and in 25-30% of intermediate grade NHL. This translocation results in the juxtaposition of the bcl-2 apoptotic inhibitor oncogene in the 18q21 chromosomal band to the heavy chain region of the immunoglobulin (Ig) locus in the 14q32 chromosomal band, which results in its overexpression. Translocation t (11; 14) (q13; q32) results in the over-expression of bcl-1 (cyclin-D1 / PRAD1), a cell cycle control gene in the band

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11q13 chromosomal, and is a diagnosis of mantle cell lymphoma. In follicular lymphomas, an increase in the frequency of chromosome gain involving chromosome 12 has been revealed, among others, by MFISH analysis (Benz et al., 1996, Horsman et al., 2001, Lestou et al., 2003), and the increase in genetic expression of the chromosomal bands with gain, which include the SAS gene in 12q13-q14 (Lestou et al., 2003) was observed. Translocation events are considered primarily responsible for the onset of FL disease, while unbalanced chromosomal gains and losses (which also reflect the patterns of genetic expression) characterize the evolution of the clones and the progression of the disease. Accordingly, it has been shown that elimination (17p) and gain / amplification of chromosome 12 correlate with an adverse clinical outcome or transformation into diffuse large cell lymphoma (DLCL) (Martinez-Climent et al., 2003, Höglund et al., 2004). Until now, however, it is unknown which gene or genes are affected by chromosome changes in chromosome 12 mentioned above.

Recently, it was shown that a new NAV3 gene supposed tumor suppressor is eliminated / translocated in most common types of cutaneous T-cell lymphoma (CTCL) (Karenko et al., 2005, WO03066898). In B lymphocyte lymphomas, aberrations have been described in Bcl-2, CD20, PAX-5, and BCL-6 but have not achieved wide clinical applications.

Therefore, new biomarkers are justified to provide more effective and early diagnoses of B-lymphoma lymphomas susceptible to targeted therapies. New markers are also needed to differentiate lymphomas.

Brief Description of the Invention

The invention is defined by the appended claims.

The object of the invention is therefore to provide new procedures and means for the diagnosis and follow-up of patients who have symptoms of B-lymphocyte lymphoma, such procedures and means allowing an early and specific diagnosis of B-lymphocyte lymphoma and identifying B-lymphocyte lymphomas. susceptible to targeted therapies.

Another object of the invention is proportional to new procedures and means that allow premature identification of patients with an increased risk of developing aggressive lymphoma and thus enable effective cancer prevention.

A further object of the invention is to provide new procedures and means for the development of new guidelines for monitoring therapeutic interventions. Also described herein, although not claimed, the development of new treatment modalities for B lymphocyte lymphomas, such procedures and means prolong the stage of disease remission and introduce new possibilities to combat the disease and for the patient recovery

Another object of the invention is to provide new methods and means for differentiating B lymphocyte lymphomas into prognosis groups indolent and aggressive forms.

The present invention relates to a method characterized by detecting genetic aberrations of the NAV3 gene in a biological sample, specifically changes in the number of copies of the NAV3 gene, indicating the presence of aberrations a lymphocyte of B lymphocytes.

The present invention further relates to a method for detecting genetic aberrations of the NAV3 gene, preferably loss or gain, enhancing the loss or gain of NAV3 differentiation of B lymphocytes into prognostic groups of indolent and aggressive lymphomas.

In a preferred embodiment of the invention genetic aberrations are determined by fluorescent in situ hybridization (FISH).

In another preferred embodiment of the invention the B lymphocyte lymphoma is a follicular lymphoma or a diffuse large B lymphocyte lymphoma.

The present invention also relates to a use of the NAV3 gene for the diagnosis or therapy of B lymphocyte lymphomas.

The present invention further relates to a use of the NAV3 gene as a biomarker for B lymphocyte lymphomas, preferably the NAV3 gene is used as a marker for the malignancy of B lymphocyte lymphoma.

The present invention opens up new possibilities in the advancement of therapies for B lymphocyte lymphomas. Classifying patients into the two groups of indolent and aggressive forms of lymphoma will allow physicians in charge of treating the patient to select individual therapeutic modalities, the most effective for That determined patient.

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With the invention of new biomarkers, such as chromosomal or genetic differences in a group with lymphoma that have the same original diagnosis, the list of different types of lymphomas will increase and provide in the future means and bases for a more specific therapy (characterized by the expression "personalized medicine").

Brief description of the drawings

The invention will now be described in more detail by means of preferred embodiments with reference to the accompanying drawings, in which

Figure 1 shows the results of NAV3-specific FISH with follicular lymphoma. Each triangle represents a case separately. Five cases were analyzed and 200 cells counted for each case. The cells were grouped as normal cells, polyploids, cells with NAV3 removed or cells with amplified NAV3. The results are shown as the percentage of each cell type. Figure 2 shows the specific FISH of NAV3 with diffuse large B lymphocyte lymphoma samples. Each triangle represents a case separately. Seven cases were analyzed and 200 cells were counted for each case. The cells were grouped as normal cells, polyploids, cells with NAV3 removed or cells with amplified NAV3. The results are shown as the percentage of each cell type.

Detailed description of the invention

The present invention is based on a method for detecting genetic aberrations in the NAV3 gene, in which aberrations are detected by deviations from the number of copies with respect to normal.

As used herein "genetic aberration" refers to deletions (losses) or amplifications (gains) of the NAV3 gene, which can be detected by the change in the number of copies of the gene.

As used herein "deletion" refers to the absence of a fragment of the NAV3 gene, a gene or a chromosomal fragment that contains the gene. In a preferred analysis procedure the elimination means less than two copies of the NAV3 signal.

As used herein the term "amplification" refers to the gain of genetic material such as a genetic fragment, a gene or a chromosomal fragment containing the gene. In a preferred analysis procedure amplification means more than two copies of the chromosome centromere.

As used herein the term "polyploid cell" refers to cells that have more than two copies of the chromosome centromere.

As used herein the term "B lymphocyte lymphoma" refers to malignant (cancerous) non-Hodgkin lymphomas produced by B lymphocytes.

NAV3 (neuronal navigator, also called POMFIL1) is a spliced gene (40 exons) located on chromosome 12q21 and expressed in brain tissue, activated T lymphocytes, placenta, colon, and certain cancer cell lines (Coy et al. 2002; Maes et al., 2002; Karenko et al. 2005). The expression of NAV3 is strongly reduced in 40% of primary tumors of neuronal or glial origin, on the other hand, it is positively regulated after a brain injury (Coy et al., 2002).

The amino acid sequence of NAV3 is well conserved between species, indicating that NAV3 has an important role in cellular processes. As expected from the amino acid sequence, NAV3 may have a role in cell signaling or tumor suppression. It also shows the properties of a helicase (enzyme that unwinds the helical structure of DNA); helicases have a role in maintaining the stability of chromosomes, and their deficiency could produce a hyper-recombination phenotype with elimination mutants, and also the loss of heterozygosity and the increased exchange of sister chromatids.

In the present invention, changes in copy number in a series of samples from the two most frequent B-lymphocyte lymphomas by fluorescent in situ hybridization (FISH) were studied using locus-specific probes. In addition to the elimination in NAV3, the number of cells with polyploidy and amplification in NAV3 was recorded. Amplification in NAV3 was found in follicular lymphoma (Figure 1), but both amplification and elimination were found in the most aggressive form of B lymphocyte lymphoma, diffuse large cell lymphoma (Figure 2). Therefore, genetic aberrations of the NAV3 gene that are characterized by the loss or gain of NAV3 are shown as a marker of the malignancy of lymphomas that originate from B lymphocytes (B cells).

According to a preferred embodiment of the present invention, the presence or absence of the NAV3 gene in a biological sample can be detected by any suitable detection method to detect the expression of a gene or the number of copies, that is, procedures that are based on the number of copies of the gene (or DNA) and / or those based on the detection of gene expression products (mRNA or protein). Such procedures are readily recognized by those skilled in the art and include conventional methods of

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polymerase chain reaction (PCR), RT-PCR, in situ hybridizations, such as FISH, matrix-CGH, in situ mRNA hybridization, high density single nucleotide polymorphism matrices (SNP), Northern analysis, analysis of Southern and Western, immunohistochemistry, and other immunoassays, such as ELISA. Preferred procedures are suitable for routine use in clinical laboratories. The most preferred is the change in the number of copies of NAV3 detected by FISH.

In the process of the invention, the biological sample can be any suitable tissue sample, such as a lymph node or bone marrow biopsy. The biological sample can be pretreated, if necessary, in a suitable manner known to those skilled in the art.

The therapeutic restoration of the normal function of the NAV3 gene is also not described here. This can be achieved by increasing the expression of functionally homologous genes, introducing an intact NAV3 gene or using an altered form of the NAV3 gene or antisense oligonucleotide against NAV3 with any currently available genetic therapy technique to prevent the progression of a proliferative disease. In particular, cell growth can be decreased or even stopped by such therapy. Such techniques include ex vivo and in situ therapy procedures, the former comprising the transduction or transfection of an altered or intact NAV3 gene (or its functional domains) in recombinant form

or peptide or as antisense oligonucleotides or in a vector to the patient, and the latter comprising the insertion of the altered gene or oligonucleotide into a carrier, which is then introduced into the patient. Depending on the disease to be treated, a temporary cure or a permanent cure can be achieved. Alternatively, monoclonal or humanized antibodies or peptides that bind to the NAV3 protein can be used.

or to the fusion gene that is generated as a result of translocation, to suppress the function of the altered NAV3 protein and in this way the growth of tumor cells can be slowed down or even stopped. Antibody against NAV3 could also be used to carry other agents, such as cytotoxic substances, to cancer cells that overexpress the NAV3 gene. Such agents could then be used to specifically destroy cancer cells.

The following examples are given for a further illustration of the invention.

Example 1

SPECIFIC FISH ANALYSIS OF NAV3 OF LYMPHOCYT B LYMPHOMES

SAMPLES

Samples for the FISH trial were prepared from 12 randomly selected cases of B lymphocyte lymphoma. Five cases represented by follicular lymphoma (FL) and seven cases of diffuse large B lymphocyte lymphoma (DLBCL). All tissue samples were so-called imprint samples, which were prepared from recent biopsy material by gently pressing the biopsy against a Super Frost Plus slide so that the cells were deposited on the slide. The imprint preparations were stored at -70 ° C.

MARKING OF THE PROBES

Two clones of the bacterial artificial chromosome (BAC) specific for NAV3 DNA (RP11-36P3 and RP11-136F16; Research Genetics Inc., Huntsville, AL, USA) were labeled with Alexa594-5-dUTP (Invitrogen) and labeled labeled the chromosome 12 centromere probe (pA12H8; American Type Cell Culture) with Alexa488-5-dUTP (Invitrogen) using nick translation (Hyytinen et al. 1994). 50-75 ng of each labeled BAC and 30 ng of the centromere probe were mixed together with 1 µg of human COT1 DNA (Invitrogen) and precipitated in sodium acetate and ethanol. The precipitated probe mixture was diluted in 10 µl of hybridization buffer (15% w / v dextran sulfate, 70% formamide in 2x SSC, pH 7.0).

FLUORESCENT HYBRIDIZATION IN SITU

The slides were fixed with 4% paraformaldehyde in PBS for 1 minute on ice. After washing with PBS, enzymatic digestion of the slides was carried out with proteinase K (Sigma; 0.66 /g / ml in 20 mM Tris-HCl, pH 7.5, 2 mM CaCl2) at +37 ° C for 6 minutes After dehydration and air drying, the probe mixture was placed with slide pipettes and the slides were denatured for 5 min at +75 ° C on a hot plate. Hybridization was carried out for 48 h at +37 ° C. The slides were washed three times with 1.5 M Urea, 0.1x SSC at +47 ° C for 10 minutes, once with 0.1 x SSC for 10 minutes at +47 ° C, followed by three washes with PBS, 0 , 1% NP-40 at room temperature. Finally, the slides were rinsed with distilled water, air dried and mounted on Vectashield Mounting Medium with 4 ’, 6-diamino-2 phenylindole dihydrochloride (DAPI; Vector).

ANALYSIS AND RESULTS

The results of the FISH were evaluated using an Olympus BX61 microscope (Tokyo, Japan) equipped with a 60x oil immersion lens and a triple bandpass filter for simultaneous detection of Alexa488, Alexa594 and DAPI (Chroma Technology Corp. , Brattleboro, VT, USA). 200 cells from each case were analyzed and

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the cells were grouped as normal if they had two markers per centromere of chromosome 12 and two for NAV3. Polyploid cells had three or more centromere markers. The elimination in NAV3 was defined when the number of centromere markers was greater than the number of the NAV3 markers and the amplification in NAV3 was defined when the number of the NAV3 markers was greater than the markers of the NAV3.

5 centromere. The analyzes were blinded for the diagnosis or sample identity by two independent analyzers. 3/5 cases of follicular lymphoma studied (60%) showed an amplification in clear NAV3. No deletion was detected in NAV3 (Figure 1). In the case of diffuse lymphomas of large B lymphocytes 1/7 (14%) of the samples showed NAV3 amplification and 1/7 (14%) of the samples showed NAV3 elimination (Figure 2).

Claims (7)

1. A procedure for the diagnosis of a B lymphocyte lymphoma, characterized by the detection of genetic aberrations of the NAV3 gene in a biological sample, indicating the presence of aberrations a B lymphocyte lymphoma. A method according to claim 1, characterized in that the genetic aberrations are changes in the number of copies of the NAV3 gene. 3. A method according to claim 2, wherein the loss or gain of NAV3 allows differentiation of B lymphocyte lymphomas into prognosis groups. 4. A method according to claim 3, wherein the prognosis groups are the indolent and aggressive forms of B lymphocyte lymphomas.

5.

A method according to any of the preceding claims, characterized in that the aberrations are determined by fluorescence in situ hybridization (FISH).

6.

A method according to any of the preceding claims, characterized in that the B lymphocyte lymphoma is a follicular lymphoma.

A method according to any of the preceding claims, characterized in that the B lymphocyte lymphoma is a diffuse lymphoma of large B lymphocytes.

8.

 The use of the NAV3 gene as a biomarker for B lymphocyte lymphomas.

9.

 The use according to claim 8, characterized in that the loss or gain of the NAV3 gene is a marker of the malignancy of B lymphocyte lymphoma.

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